1. Field of the Invention
The present invention relates to a biocontrol formulation for delivering non-toxigenic strains of fungi, such as for example, Aspergillus including Aspergillus flavus (A. flavus), Aspergillus parasiticus (A. parasiticus), Aspergillus oryzae (A. oryzae), and Aspergillus sojae (A. sojae) to crops in methods for the control of toxin contamination in agricultural commodities. The present invention also relates to a method for preparing the biocontrol formulations. This invention claims priority of United States Provisional Patent Application 60/145,251 filed Jul. 26, 1999; which is herein incorporated by reference.
2. Description of the Related Art
Aflatoxins are potent hepatotoxic, carcinogenic compounds produced by A. flavus Link:Fr. and A. parasiticus Speare (CAST, In: Mycotoxins: Economic and Health Risks. Report 116, 99 pp., Council for Agricultural Science and Technology, 137 Lynn Avenue, Ames, IA 50010). Cyclopiazonic acid (CPA) is another potent mycotoxin that is produced by A. flavus, but not by A. parasiticus. When these fungi invade and grow in commodities such as peanuts, corn, cottonseed, and tree nuts, the resulting contamination with the aflatoxins and CPA often makes the commodity unfit for consumption. The United States peanut industry has identified aflatoxin contamination of peanuts as the number one problem for which a solution is needed (Consensus Report of the National Peanut Council Quality Task Force, 1987, National Peanut Council, Alexandria, Va. 22314). Because peanuts are used primarily for food, strict regulatory limits for the amount of aflatoxin allowable in finished peanut products have been established. Although the United States Food and Drug Administration has an action level of 20 ppb of total aflatoxins in food products, international tolerances for aflatoxin are much lower, typically in the range of 0-4 ppb, and are important because U. S. companies compete internationally in the market to export peanuts and peanut products. For this reason the United States peanut industry has a goal to ensure the delivery of aflatoxin-free peanut products by the year 2000. Although aflatoxin contamination of peanuts can occur during postharvest curing and storage, the most significant contamination usually occurs prior to harvest during periods of late-season drought stress as peanuts are maturing. The only known method for controlling preharvest aflatoxin contamination in peanuts is irrigation, an option that is unavailable to the majority of peanut growers.
Cyclopiazonic acid is an indole-tetramic acid that was first isolated from cultures of Penicillium cyclopium Westling in 1968 (Holzapfel, Tetrahedron,Volume 24, 2101-2119, 1968). CPA is now know to be produced by a variety of fungi including P. patulum, P. viridicatum, P. puberulum, P. crustosum, P. cainemberti, A. flavus, A. versicolor and A. oryzae. In addition, CPA has been found as a natural contaminant of corn and peanuts, often occurring together with aflatoxin (Lansden and Davidson, Applied and Environmental Microbiology, Volume 45, 766-769,1983; Urano et al., Journal of AOAC International, Volume 7S, 838-841, 1992). It was also implicated as the causative agent in a human intoxication involving consumption of contaminated millet (Rao and Husain, Mycopathologia, Volume 89, 177-180, 1985). With the discovery of CPA production by A. flavus , 54 isolates of A. flavus were investigated for production of CPA and aflatoxin (Gallagher et al., Mycopathologia, Volume 66, 31-36, 1978). It was found that 28 of the 54 (52%) produced CPA whereas only 18 (33%) produced aflatoxin. Regulatory limits for CPA have not been established; however, because of the co-occurrence of aflatoxin and CPA in commodities, efforts to attain biological control of aflatoxin also need to attain control of CPA.
It has been previously found that co-cultivation of either A. parasiticus or A. flavus with species of Penicillium reduce levels of aflatoxin production while co-cultivation of Fusarium species had no such effect (Ehrlich et al., Experiential, Volume 41, 691-693, 1985). These tests did not involve the use of a soil environment. Co-cultivation with A. niger completely eliminated the production of aflatoxin by a culture of A. flavus (Wicklow et al., Phytopathology, Volume 70, 761-764, 1980). This testing was done under laboratory controlled conditions in which the food source involved sterilized corn.
The conventional method for producing biocontrol formulations for aflatoxin control is via solid state fermentation (SSF) using a suitable substrate such as wheat or rice (Bock and Cotty, Biocontrol Science and Technology, Volume 9, 529-543, 1999). It is necessary to rehydrate, sterilize, inoculate and ferment the substrate with the biocontrol organism(s) and dry the formulation to a safe moisture level. Scale-up of the SSF method is difficult since manufacturing facilities are currently not available. The SSF method is not cost effective and also has potential safety problems since some spores are produced that can become airborne during manufacture and field application.
Cotty (U.S. Pat. No. 5,171,686-Dec. 15, 1992 and U.S. Pat. No. 5,294,442-Mar. 15, 1994) discloses a non-toxigenic strain of A. flavus which inhibits aflatoxin production by toxigenic strains. Formulations for field delivery were prepared using the SSF method. The patent teaches that agricultural commodities inoculated simultaneously with both a non-toxigenic strain and a toxigenic strain produce seed with up to 100-fold less aflatoxin than commodities inoculated with a toxigenic strain alone.
Cole et al.(U.S. Pat. No. 5,292,661-Mar. 8, 1994) and Dorner et al.(Journal of Food Protection, Volume 55, 888-892, 1992) disclose a non-aflatoxigenic strain of A. parasiticus. The biocompetitive agent was prepared by growing the strains in 2.8 liter Fernbach flasks on liquid YES medium containing 15% sucrose, 5% mycological broth, pH 4.8, and 2% yeast extract; for two weeks at 27.degree. C. Contents of the flasks were combined and homogenized in water plus 0.05% Tween 20. The homogenate was strained and applied over rows of peanuts using a garden sprinkler. The references teach the use of this strain as a biocontrol agent which reduces aflatoxin contamination of soilborne crops.
U.S. Pat. No. 5,730,973 (Morales et al., Mar. 24, 1998) discloses a biocontrol formulation containing 5-8% by weight of spores or active units of a fungus, 5-60% by weight of one or more suitable wetting agents and dispersants, 2-20% by weight of at least one protective substance which prevents desiccation which can include vegetable oil, 5-70% by weight of magnesium silicate or aluminum silicate, 0.5-20% by weight of at least one substance which protects against UV radiation, and a residual amount of water content at about 2-10%. The formulation is water dispersable and useful for the control of insect pests.
U.S. Pat. No. 5,413,784 (Wright et al., May 9, 1995) discloses a biopesticide which includes entomophathogenic fungi having virulence against targeted insect pests, an arrestant and feeding stimulant and optionally a pheromone. The feeding stimulant is defined as a combination of protein, carbohydrates, and lipid oil which can be formulated as a dispersable granule.
While various biocontrol formulations for control of toxigenic fungi are known in the art, there still remains a need for an effective biocontrol formulation for controlling toxigenic fungi. The present invention described below includes formulations and methods of preparing formulations that includes non-toxigenic strains of fungi which displace toxigenic fungi. The present invention also provides a method for controlling toxigenic fungi in agricultural crops which is different from the related art biocontrol methods